A reversed-phase high-performance liquid chromatography (RP-HPLC) technique is described that can be used for the determination of niferidil in rat blood serum. The RP-HPLC procedure is carried out on a Silasorb SF C8 column (150 × 4 mm, 5 μm) eluted with a MeOH:H2 O (45:55) mobile phase (adjusted to pH 2.5 with orthophosphoric acid and then to pH 3.5 with propylamine) at a flow rate of 1 mL/min with UV detection at 265 nm. The procedure is as follows: mix rat blood serum (1 mL), NaHCO3 solution (100 μL), and CHCl3 (3 mL) in a 10-mL glass centrifuge tube. Shake for 2 min and centrifuge at 3,000 rpm for 10 min. Transfer the CHCl3 phase into another tube and evaporate to dryness under a stream of nitrogen. Reconstitute the residue in mobile phase (100 μL) and inject an aliquot (20 μL) into the chromatographic column. The average recovery of niferidil is 70%. The detection limit is 0.01 μg/mL. The method was linear from 0.02 to 0.5 μg/mL with a correlation coefficient of 0.968. The coefficient of variation was less than 12% within a day (n = 5). The proposed method was successfully used to determine some pharmacokinetic parameters after intravenous injection of niferidil in rats.